Throughout this application, various publications are referred to. These publications are incorporated herein in their entireties and constitute part of the description.
Arthritis is a group of conditions involving damage to the joints of the body and is the leading cause of disability in people older than fifty-five years. The term comes from the Greek ‘arthros’, meaning joint, and ‘itis’, meaning inflammation.
There are different forms of arthritis that may be generally grouped into two main categories, inflammatory arthritis, and degenerative arthritis, each has a different causes. One critical assessment of patients involves therefore differentiating inflammatory arthritis from a degenerative process, particularly, since the treatment approaches are completely different.
Inflammatory arthritis is characterized by synovitis, bone erosions, osteopenia, soft-tissue swelling, and uniform joint space narrowing. Degenerative or mechanical arthritis, known generally as osteoarthritis, are group of conditions characterized by cartilage damage. More specifically, in addition to lack of the findings described for inflammatory joint disease, degenerative findings include osteophyte formation and bone sclerosis.
Inflammatory arthritis may be further divided into several subgroups. Involvement of a single joint is indicative of either gout or a septic arthritis caused by joint infection, usually, staphylococcal, streptococcal or gonococcal infection. A systemic arthritis, in contrast, is characterized by involvement of multiple joints, and includes two main categories, rheumatoid arthritis and seronegative spondyloarthropathy.
Rheumatoid arthritis is most common in women aged 30-60 years. Serologic markers such as rheumatoid factor and antibodies to cyclic citrullinated peptide are important indicators of rheumatoid arthritis. The radiographic features of rheumatoid arthritis are those of joint inflammation and include particular osteopenia, uniform joint space loss, bone erosions, and soft-tissue swelling. Because of the chronic nature of the inflammation, additional findings such as joint subluxation and subchondral cysts may also be evident.
The seronegative spondyloarthropathy category includes psoriatic arthritis, reactive arthritis, and ankylosing Spondylitis, and is characterized by signs of joint inflammation, multiple joint involvement, and distal involvement in the hands and feet with added features of synovial proliferation. These features accompany the specific features of these arthritic diseases i.e. Psoriatic skin diseases in psoriasis, recent infection in reactive arthritis and spinal and sacroiliac involvement in ankylosing spodylitis.
Adjuvant Arthritis (AA) is a well established experimental model of inflammatory and autoimmune arthritis which can be induced in susceptible strains of rats such as inbred Lewis or Wistar strains upon vaccination with heat-killed Mycobacterium Tuberculosis (MT) in complete Freund's Adjuvant (CFA) [Pearson, C.M., Proc. Soc. Exp. Biol. Med. 91:95-101 (1956); Pearson, C.M. & Wood, F.D., Arthritis Rheum. 2:440 (1959); Waksman, B.H. and Wennersten, C., Int. Arch. Allergy 23:129 (1963)]. The disease cannot be induced in resistant strains of rats (e.g., Brown-Norway; Fisher [Hogervorst, E.J.M., et al. Eur. J. Immunol. 21:1289-1296 (1991); Griffiths, M.M., et al., Arthritis Rheum. 36:254 (1993)], and Lewis rats develop resistance to re-induction of the disease after recovery from arthritis.
Heat shock proteins are a family of highly conserved proteins. There is ˜50% amino acid identity between the Mycobacterial HSP 65 and the mammalian
mammalian HSP 60 [Jindal, S., et al., Mol. Cell. Biol. 9:2279-2283 (1989)]. The role of the 65KD heat shock protein (HSP 65) of MT in the pathogenesis of autoimmune arthritis, both in experimental animals [Van Eden, W., et al., Nature (Lond.) 331:171-173 (1988); Holoshitz, J., et al., Science (Wash. DC) 219:56-58 (1983)] as well as in humans [Holoshitz, J., et al., Lancet 2:305-309 (1986); Res, P.C.M., et al., Lancet 2:478-480 (1988); Gaston, J.S.H., et al., J. Immunol. 143:2494-2500 ( 1989)], has been investigated intensively in the past several years. For example, Barker et al. [Barker et al., Autoimmunity 14:73-77 (1992)] describe the suppression of arthritogenic immune responses in mice given HSP 65 and pristane. The antigen used to elicit the response was full-length HSP 65, and no attempt was made to investigate the effect of specific sub-domains or peptides deriving from this protein.
Resistance to adjuvant arthritis can be conferred by several factors: genetic background (e.g. Black-Norway or Fisher strains), old age, previous disease and pre-immunization of susceptible rats with mycobacterial HSP65. Evidence has been reported that protection from disease may be due to cellular responses to HSP65 [Hogervorst (1991) ibid.; Thole, J. et al. Infect. Immun. 55:1466-1475 (1987); Lider, O. et al. Proc. Natl. Acad. Sci. 84:4577-4580 (1987); Billingham, M. et al. J. Exp. Med. 171:339-344 (1990); Moudgil, K. et al. J. Exp. Med. 185:1307-1316 (1997)] suggesting that this protein contains different epitopes which participate in both pathogenesis and acquisition of resistance. The inventors have previously shown that resistance to AA can be transferred to a susceptible strain of rats by intravenous infusion of immunoglobulins from resistant strains, and that resistance is associated with the presence of antibodies against the 65KD MT heat shock protein (HSP 65) [Ulmansky, R., and Y. Naparstek, Eur. J. Immunol. 25:952-957 (1995)].
The present invention now illustrate the fine epitope specificity of the anti-HSP antibodies of arthritis—susceptible and resistant rats. As shown by the invention, naive Lewis rats lack antibodies to certain epitopes of the mycobacterial HSP 65 which are found naturally in young BN and old naive Lewis rats, and that are acquired by young Lewis rats after recovery from the disease. Analysis of the primary and tertiary structure of the whole MT HSP 65KD molecule indicated that these “protective” epitopes are potential B-cell epitopes with a non-conserved amino acid sequences that are found on the outer surface of the molecule.
Pre-immunization of Lewis rats with one of the “protective” epitopes prior to induction of the disease induced antibodies against the whole molecule as well as resistance to disease induction. This sixteen amino acid peptide, termed peptide-6 (also denoted by SEQ ID: NO. 2) corresponds also to the self-HSP 60 epitope to which antibodies were found in the arthritis resistant rats, but not in the arthritis-susceptible naive Lewis rats.
The present invention therefore provides a working hypothesis centered on the failure of the immune system of RA susceptible patients to produce an antibody against this B-cell epitope peptide-6. The invention further shows that antibodies directed against peptide-6 interact not only with peptide-6, but moreover, they cross react directly with a surface ligand on macrophages, and this interaction is the key to the mechanism of action of these antibodies. Following binding of the anti-peptide-6 antibodies to macrophages, there is activation of a signal transduction pathway that leads to an increase in production and secretion of cytokines, specifically IL-10 that as an anti-inflammatory cytokine, attenuates and inhibits an inflammatory process, thereby leading to amelioration and treatment of an inflammatory disorder. This tilts the balance between pro-inflammatory Th1 cytokines, such as tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory Th2 cytokines, such as IL-10. Modulation of the Th1/Th2 balance towards a Th2 anti-inflammatory response by the antibodies of the invention may be therefore applied for treating inflammatory disorders. The results disclosed by the present invention clearly establish the feasibility of using the B-cell epitope peptides of the invention and particularly the use of antibodies directed against such peptides as immuno-modulators, specifically for modulating the Th1/Th2 balance towards a Th2 anti-inflammatory response.
Therefore, one object of the invention is to provide peptides, specifically peptides having the amino acid sequence of any one of SEQ ID NO. 1, 2, 3, and 4, antibodies directed against said peptides as well as compositions comprising the same for the treatment of immune-related disorders, particularly, inflammatory and autoimmune disorders.
Another object of the invention is to provide methods and compositions for enhancing the expression of IL-10 in a subject in need thereof.
These and other objects of the invention will become apparent as the description proceeds.